A small molecule that inhibits the activity of REXO4 R-loop-dissolving enzyme and its application in the preparation of tumor drugs

Abstract

The present invention relates to the field of biomedicine and discloses a small molecule that inhibits the activity of the REXO4 enzyme that degrades R-loops and its use in the preparation of an anticancer drug. The small molecule obtained by the present invention can inhibit the activity of the REXO4 enzyme that degrades R-loops, thereby increasing the content of R-loop structures in tumors and achieving the purpose of treating tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a small molecule that inhibits the activity of REXO4 R-loop-dissolving enzyme and its application in the preparation of tumor drugs. Background Art

[0002] R-loop structures are a common structure that occurs during RNA transcription. They consist of a free DNA strand and an RNA-DNA hybrid strand. Transcriptional replication occurs frequently in dividing cells, particularly tumor cells. During RNA transcription, when RNA polymerase encounters obstacles, R-loop structures may form.

[0003] While R-loop structures serve as important regulators under physiological conditions, such as those regulating gene expression, CSR in B cells, and T cell receptor reprogramming, abnormal accumulation of R-loop structures can block transcription or expose single-stranded DNA to nucleases. During S phase of the cell cycle, transcription-replication collisions between DNA replication forks and transcription complexes are the most common instances of R-loop-mediated DNA damage. These collisions can lead to replication arrest or even replication fork collapse, resulting in DNA double-strand breaks and posing a risk to genomic stability.

[0004] To mitigate the potentially toxic consequences of R-loop structures, cells have evolved a variety of regulatory mechanisms to prevent their formation or resolve them. For example, the endonuclease RNase H1 (RNASEH1) can specifically degrade the RNA strands of R-loops, allowing the DNA strands to reanneal. Tumor cells face the risk of DNA damage caused by high replication and transcription, and replication stress is the primary source of endogenous DNA damage in tumor cells. If accumulated transcription-replication collisions in tumor cells are not effectively alleviated, they can enter a state of mitotic collapse, leading to cell death.

[0005] Therefore, targeted degradation of enzymes related to R-loop structure has important biological and clinical significance for tumor treatment. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a small molecule that inhibits the activity of REXO4 enzyme to digest R-loop and its use in the preparation of tumor drugs. The small molecule obtained by the present invention can inhibit the activity of REXO4 enzyme, thereby promoting the accumulation of R-loop structure in tumors and achieving the purpose of treating tumors.

[0007] The present invention provides a small molecule that inhibits the activity of REXO4 in digesting R-loop enzymes. The structural formula of the small molecule is:

[0008] .

[0009] The present invention also provides the use of the small molecule that inhibits the activity of REXO4 in clearing the R-loop enzyme in the preparation of drugs for treating tumors.

[0010] Furthermore, the tumor includes one or more of human head and neck squamous cell carcinoma and oral squamous cell carcinoma.

[0011] The present invention also provides the use of the small molecule that inhibits the REXO4 R-loop digestion enzyme activity in the preparation of a drug that inhibits the REXO4 enzyme activity.

[0012] The present invention also provides the use of the small molecule that inhibits the REXO4 R-loop digestion enzyme activity in the preparation of a drug that promotes the accumulation of R-loop structures in tumor cells.

[0013] The embodiments of the present invention have the following technical effects:

[0014] 1. The small molecules obtained by the present invention can inhibit the activity of REXO4 enzyme that digests R-loop structure, thereby promoting the accumulation of R-loop structure in tumors and achieving the purpose of treating tumors.

[0015] 2. The small molecules obtained by the method of the present invention can inhibit the activity of REXO4 to eliminate R-loop enzymes, increase the level of R-loop structure in UM-SCC1 cells and UM-SCC25 cells, and achieve the purpose of treating human head and neck squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is the mass spectrum of the small molecule of Example 1 of the present invention.

[0018] Figure 2 This is the preparation process of the small molecule obtained in Example 1 of the present invention.

[0019] Figure 3 is the test result of Example 2 of the present invention, wherein Figure 3 A in the middle is the cleavage preference of REXO4 enzyme; Figure 3 Middle B is a typical graph and statistical graph of the in vitro cleavage efficiency of R-loop hybrid chains by REXO4 enzyme; Figure 3Middle C is a gel image of His-REXO4 protein stained with Coomassie brilliant blue.

[0020] Figure 4 is the test result of Example 3 of the present invention, wherein Figure 4 Middle A is the cleavage efficiency of REXO4 on RNA in RNA-DNA hybrid chains at different iR4 concentrations; Figure 4 Middle B is a statistical graph of the RNA cleavage efficiency of REXO4 in RNA-DNA hybrid chains at different iR4 concentrations.

[0021] Figure 5 is the test result of Example 4 of the present invention, wherein Figure 5 Middle A shows UM-SCC1 cells treated with different doses of iR4 and GFP-dRH1 was detected; Figure 5 Middle B is a statistical graph of the relative fluorescence intensity of GFP in UM-SCC1 cells; Figure 5 Middle C shows UM-SCC25 cells treated with different doses of iR4 and GFP-dRH1 was detected; Figure 5 Middle D is a statistical graph of the relative fluorescence intensity of GFP in UM-SCC25 cells.

[0022] Figure 6 is the test result of Example 5 of the present invention, wherein Figure 6 Middle A is the detection of GFP-dRH1 in UM-SCC1 cells that can express RnaseH1 protein and those that cannot express RnaseH1 protein by treatment with or without iR4; Figure 6 Middle B is a statistical graph of the relative fluorescence intensity of GFP in UM-SCC1 cells; Figure 6 Middle C is the detection of GFP-dRH1 in UM-SCC25 cells that can express RnaseH1 protein and those that cannot express RnaseH1 protein by treatment with or without iR4; Figure 6 Middle D is a statistical graph of the relative fluorescence intensity of GFP in UM-SCC25 cells.

[0023] Figure 7 is the test result of Example 6 of the present invention, wherein Figure 7 Middle A is a mouse tumor-bearing model and typical tumor images and statistical graphs; Figure 7 Middle B is GFP-dRH1 tissue immunofluorescence staining and statistical graph; Figure 7 Middle C is γH2AX tissue immunofluorescence staining and statistical graph; Figure 7 Middle D is Cxcl10 tissue immunofluorescence staining and statistical graph, Figure 7 Middle E is AcCasp3 tissue immunofluorescence staining and statistical graph. DETAILED DESCRIPTION

[0024] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0025] In a first aspect, some embodiments of the present invention provide a small molecule that inhibits the activity of REXO4 in clearing the R-loop enzyme, wherein the structural formula of the small molecule is:

[0026] .

[0027] In a second aspect, some embodiments of the present invention also provide the use of the small molecule that inhibits the activity of REXO4 to eliminate R-loop enzyme in the preparation of drugs for treating tumors.

[0028] In some embodiments, the tumor comprises one or more of human head and neck squamous cell carcinoma and oral squamous cell carcinoma.

[0029] In a third aspect, some embodiments of the present invention further provide the use of the small molecule that inhibits the activity of REXO4 enzyme in clearing R-loop in the preparation of a drug that inhibits the activity of REXO4 enzyme in clearing R-loop.

[0030] In a fourth aspect, some embodiments of the present invention further provide the use of the small molecules that inhibit the activity of REXO4 to eliminate R-loop enzymes in the preparation of drugs that promote the accumulation of R-loop structures in tumor cells.

[0031] The following is elaborated with reference to specific embodiments:

[0032] Example 1:

[0033] , recorded as: iR4.

[0034] Example 1 Product preparation was commissioned to Tianjin WuXi AppTec New Drug Development Co., Ltd. The preparation process is as follows Figure 2 shown.

[0035] NMR data:

[0036] 1HNMR:EC29906-10-P1N (400 MHz, DMSO- d 6) δ ppm = 10.36 (s, 1H), 8.38(d, J = 7.6 Hz, 1H), 8.15 (d, J= 8.0 Hz, 1H), 7.92 (d, J = 7.6 Hz, 1H), 7.76-7.64(m, 4H), 7.54-7.42 (m, 4H), 7.34-7.28 (m, 2H), 7.26-7.19 (m, 3H), 5.61 (s,2H), 4.47 (s, 2H).

[0037] The mass spectrum data of the product of Example 1 is as follows Figure 1 shown.

[0038] Example 2: Verify that REXO4 enzyme can specifically degrade RNA in R-loop structure. Different substrates (such as ssRNA, RNA-DNA, etc.) were used to test the cleavage preference of REXO4 enzyme. The results are as follows: Figure 3 As shown in Figure A, it can be found that the in vitro enzyme activity experiment verified that the REXO4 enzyme can specifically cut RNA in the RNA-DNA hybrid chain. Figure 3 Figure B further verified that REXO4 achieves the purpose of eliminating the R-loop structure by degrading the RNA in the simulated R-loop structure in the human body. Figure 3 Middle C is the REXO4 protein staining image used in the in vitro enzyme activity detection experiment.

[0039] Example 3: Validation of small molecule inhibitor iR4 (C 28 H 21 N5OS) can inhibit the exonuclease activity of REXO4, and the results are as follows Figure 4 As shown. Figure 4 As shown in Figures A and B, iR4 inhibits REXO4's R-loop-digesting enzyme activity in a concentration-dependent manner. As the concentration of iR4 increases, the small molecule's inhibition of REXO4 activity becomes more pronounced, resulting in a decrease in the RNA degradation rate within the RNA-DNA hybrid. Furthermore, the half-inhibitory concentration (IC50) of iR4 on REXO4 activity is approximately 1 μM.

[0040] Example 4: To further verify that iR4 can inhibit the enzymatic activity of REXO4 at the cellular level, tumor cells were stained with GFP-dRH1. The results are as follows: Figure 5 As shown. UM-SCC1 cells and UM-SCC25 cells (both UM-SCC1 cells and UM-SCC25 cells are human head and neck squamous cell carcinoma cells) were treated with different concentrations of iR4. Figure 5 Middle AB and Figure 5CD showed that iR4 can specifically enhance the accumulation of R-loop structures in tumor cells. GFP-dRH1 is a recombinant protein expressed and purified in BL21 E. coli. It consists of a GFP tag and an RNH1 D210N mutant. It is mainly used to study the regulatory mechanism of RNA-DNA hybrids (R-loops). Therefore, it can mark R-loop structures in tumor cells after cell staining. Figure 5 As can be seen in the results, with increasing iR4 concentration, the level of R-loop structures in UM-SCC1 and UM-SCC25 cells first increased and then stabilized. Therefore, the further preferred iR4 concentration is 5 μM.

[0041] Example 5: To further verify the specificity of iR4 targeting the REXO4 enzyme, SCC1 and SCC25 cells were infected with lentivirus containing the pLVX-Tet-On-Advanced (Neo) expression plasmid (purchased from Guangzhou Youbao Biotechnology Co., Ltd., Catalog No. VT1467). Stable SCC1-TR and SCC25-TR lines were selected by G418 geneticin. Stable lines selected by G418 geneticin were then infected with lentivirus containing the pLVX-Tight-puro-NLS-RNaseH1-V5-FLAG plasmid (Clontech, Catalog No. 632184). Stable cell lines that can be inducibly expressed by doxycycline (Dox)-induced ribonuclease H1 (RNaseH1) were selected by puromycin to obtain RNaseH1, an endonuclease that specifically degrades RNA in RNA-DNA hybrids. Figure 6 In the experiment, a solution containing iR4 was used as the experimental group (iR4 was dissolved in 10% v / v DMSO, 40% v / v PEG300, 5% v / v TWEEN80, and the balance was PBS buffer solution, with a concentration of 5 μM), denoted as iR4; while a solution without iR4 was used as the control group (10% v / v DMSO, 40% v / v PEG300, 5% v / v TWEEN80, and the balance was PBS buffer solution), denoted as Vehicle. Figure 6In a study, researchers found that when iR4 (indicated by "+") was added to a stable cell line expressing RNaseH1 (denoted by "Dox+") or a stable cell line not expressing RNaseH1 (denoted by "Dox-"), the GFP fluorescence intensity in SCC1 cells expressing RNaseH1 was significantly lower than that in SCC1 cells not expressing RNaseH1. Furthermore, the GFP fluorescence intensity in SCC1 cells not expressing RNaseH1 was significantly higher than that in SCC1 cells treated with a solution without iR4 (denoted by "-"). The GFP fluorescence intensity in SCC1 cells treated with a solution without iR4 did not change, and similar results were confirmed in SCC25 cells. This suggests that iR4 specifically targets REXO4, leading to the accumulation of R-loop structures, which are partially eliminated by Dox-induced RNaseH1 expression.

[0042] Example 6: To establish an animal model, 16 6-week-old C57BL / 6 mice (fully immunized mice) were randomly divided into two groups, and each mouse was subcutaneously injected with 2.5×10 6 MOC1 cells (oral squamous cell carcinoma cells) were injected. Five days after injection, the largest tumor in the mouse grew to about 100 mm. 3 Two groups of mice were treated every two days. The experimental group received a 10 mg / kg injection of a solution containing iR4 (the iR4 dose was calculated based on mouse body weight and injection volume, and then iR4 was dispersed in 100 μL of a solvent consisting of 10% v / v DMSO, 40% v / v PEG300, 5% v / v TWEEN80, and the balance PBS buffer). This group served as the experimental group, designated iR4. Another group of mice received an equal volume of a solvent (10% v / v DMSO, 40% v / v PEG300, 5% v / v TWEEN80, and the balance PBS buffer) at the same time points and served as the control group, designated Vehicle. Tumor growth and other indicators were measured after 36 days of feeding. Figure 7 A in the middle is a photo of tumor cells in mice after 36 days of feeding. It can be seen that the tumors of mice injected with iR4 are significantly smaller than those of the control group. Figure 7 Middle B is the result of staining the nucleus of tumor cells, cytokeratin 14 and GFP-dRH1, which are specific markers for head and neck squamous cell carcinoma cells. Among them, K14 is present in tumor cells, which can verify that both the control group and the iR4 group have formed tumor cells. GFP-dRH1 staining is used to stain the R-loop structure in tumor cells. Figure 7 In Figure B, it can be found that the content of R-loop structure in tumor cells of the experimental group increased, which shows that iR4 can promote the accumulation of R-loop structure in tumors. Figure 7In the middle C, the γH2AX protein of tumor cells was stained. The γH2AX protein can reflect the DNA damage of tumor cells. Figure 7 It can be found in Figure C that the DNA damage of tumor cells in the experimental group was significantly higher than that in the control group. It can be seen that iR4 can promote DNA damage in tumor cells and cause genomic instability. When the DNA of tumor cells is damaged, more cytoplasmic DNA will be produced. Cytoplasmic DNA can activate the expression of type I interferon genes. The production of type I interferon can cause the recruitment of anti-tumor related immune cells to kill tumor cells. The chemokine Cxcl10 is one of the proteins in the signaling pathway of type I interferon gene expression. Figure 7 In Figure D, the expression of Cxcl10 in the experimental group was significantly higher than that in the control group. This shows that iR4 in the present invention can enhance the expression of type I interferon gene chemokines, thereby achieving the recruitment of anti-tumor related immune cells. The tumor cell apoptosis-related marker AcCasp3 was stained, and the results were as follows: Figure 7 As shown in Figure E, AcCasp3 can promote the apoptosis of tumor cells. It can be found that the fluorescence intensity of AcCasp3 in the experimental group is significantly higher than that in the control group. This shows that iR4 in the present invention can promote the apoptosis of tumor cells through anti-tumor immunity.

[0043] In summary, the small molecules obtained in the present invention can inhibit the activity of REXO4 in digesting R-loop enzymes, thereby promoting the accumulation of R-loop structures in tumors and achieving the purpose of treating tumors.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention.

Claims

1. Use of a small molecule that inhibits the activity of REXO4 R-loop-dissolving enzyme in the preparation of a drug for treating tumors; The tumor is a human head and neck squamous cell carcinoma; The small molecule increases the level of R-loop structure in UM-SCC1 cells and UM-SCC25 cells by inhibiting the R-loop-dissolving enzyme activity of REXO4; The structural formula of the small molecule is .

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